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p62/SQSTM1-induced caspase-8 aggresomes are essential forionizing radiation-mediated apoptosisSu Hyun Lee 1,2,9, Won Jin Cho1,9, Abdo J. Najy1, Allen-Dexter Saliganan1, Tri Pham1, Joseph Rakowski3,4, Brian Loughery3,Chang Hoon Ji2,5, Wael Sakr1, Seongho Kim3, Ikuko Kato3, Weon Kuu Chung6, Harold E. Kim3,4, Yong Tae Kwon 2,5,7,8✉ andHyeong-Reh C. Kim 1,3✉

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The autophagy–lysosome pathway and apoptosis constitute vital determinants of cell fate and engage in a complex interplay inboth physiological and pathological conditions. Central to this interplay is the archetypal autophagic cargo adaptor p62/SQSTM1/Sequestosome-1 which mediates both cell survival and endoplasmic reticulum stress-induced apoptosis via aggregation ofubiquitinated caspase-8. Here, we investigated the role of p62-mediated apoptosis in head and neck squamous cell carcinoma(HNSCC), which can be divided into two groups based on human papillomavirus (HPV) infection status. We show that increasedautophagic flux and defective apoptosis are associated with radioresistance in HPV(-) HNSCC, whereas HPV(+) HNSCC fail to induceautophagic flux and readily undergo apoptotic cell death upon radiation treatments. The degree of radioresistance and tumorprogression of HPV(-) HNSCC respectively correlated with autophagic activity and cytosolic levels of p62. Pharmacological activationof the p62-ZZ domain using small molecule ligands sensitized radioresistant HPV(-) HNSCC cells to ionizing radiation by facilitatingp62 self-polymerization and sequestration of cargoes leading to apoptosis. The self-polymerizing activity of p62 was identified asthe essential mechanism by which ubiquitinated caspase-8 is sequestered into aggresome-like structures, without which irradiationfails to induce apoptosis in HNSCC. Our results suggest that harnessing p62-dependent sequestration of ubiquitinated caspase-8provides a novel therapeutic avenue in patients with radioresistant tumors.

Cell Death and Disease (2021) 12:997 ; https://doi.org/10.1038/s41419-021-04301-7

INTRODUCTIONCrosstalk and interplay among programmed cell death and cellsurvival pathways play a critical role in normal cellular processes aswell as under pathological conditions [1–4]. While apoptosis ismost responsible for programmed cell death induced bydevelopmental cues, intracellular damage or external deathstimuli, autophagy is a major cell survival pathway that removesdamaged cellular components and supports cellular metabolism[5, 6]. Autophagy has dual effects on cancer development andprogression. It exerts tumor suppressive functions by removingdamaged organelles and pathogens, contributing to the main-tenance of genomic stability in normal cells [7]. However, ascancers progress, autophagy promotes cancer cell survival,thereby exerting oncogenic activity for cancer progression andtherapy-resistance [8, 9]. Given that the impairment of the delicateinterplay between autophagy and apoptosis is a contributingfactor in the pathogenesis of many human diseases includingcancers, efforts have been made to find the molecular andfunctional points of interactions between autophagy and

apoptosis [10, 11]. In preclinical studies, pharmacological inhibi-tion or genetic manipulation of autophagic regulators such asautophagy-related proteins (ATGs), mammalian target of rapamy-cin (mTOR), and phosphatidylinositol 3-kinase catalytic subunittype 3 (PI3KC3) have been shown to sensitize tumor cells tochemotherapy-induced cell death [9, 12, 13]. These studies haveled to multiple clinical trials using autophagy inhibitors such aschloroquine or hydroxychloroquine as a strategy to promote celldeath. Although this approach showed minor improvements inclinical trials, there are concerns about specificity and side effectsassociated with the systemic inhibition of autophagy [13, 14].Thus, a critical need persists for the development of disease-specific and mechanism-based therapeutic tools to manipulateautophagic cell survival and programmed cell death.The N-degron pathway is a proteolytic system wherein single

N-terminal amino acids of proteins function as degradationdeterminants, called N-degrons, which are recognized by therecognition components (N-recognins) [15, 16]. The N-degronsinclude Arg, Lys, His (type 1; positively charged), Phe, Tyr, Trp, Leu,

Received: 8 February 2021 Revised: 3 September 2021 Accepted: 7 October 2021

1Department of Pathology, Wayne State University School of Medicine, Karmanos Cancer Institute, Detroit, MI 48201, USA. 2Cellular Degradation Biology Research Center andDepartment of Biomedical Sciences, College of Medicine, Seoul National University, Seoul 03080, Republic of Korea. 3Department of Oncology, Wayne State University School ofMedicine, Karmanos Cancer Institute, Detroit, MI 48201, USA. 4Division of Radiation Oncology, Wayne State University School of Medicine, Karmanos Cancer Institute, Detroit, MI48201, USA. 5AUTOTAC Bio Inc., Changkkyunggung-ro 254, Jongno-gu, Seoul 03080, Korea. 6Department of Radiation Oncology, Kyung Hee University Hospital at Gangdong,College of Medicine, Kyung Hee University, Seoul, Korea. 7SNU Dementia Research Center, College of Medicine, Seoul National University, Seoul 03080, Republic of Korea.8Ischemic/Hypoxic Disease Institute, College of Medicine, Seoul National University, Seoul 03080, Republic of Korea. 9These authors contributed equally: Su Hyun Lee, WonJin Cho. ✉email: yok5@snu.ac.kr; hrckim@med.wayne.eduEdited by Professor Gian Maria Fimia

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and Ile (type 2; bulky hydrophobic), as well as ATE1-mediatedarginylation-permissive residues, exposed at the protein termini[17, 18]. The human genome encodes a set of N-recognins (UBR1,UBR2, UBR4, and UBR5) whose UBR-box domains bind N-degronsfor substrate ubiquitination and proteasomal degradation [19, 20].Recently, we found that the N-degron pathway also mediatesautophagic proteolysis where the autophagic receptor p62/SQSTM1 acts as an N-recognin that binds N-degrons [21, 22]. Inthis process, the Nt-Arg residue of N-degrons binds the p62 ZZdomain to induce p62 self-polymerization and interaction withLC3 on autophagic membranes, leading to lysosomal degradationof p62-cargo complexes [21–23].Paradoxical to the well-known function of p62 as an autophagic

adaptor for cell survival, endoplasmic reticulum (ER) stress-induced apoptotic cell death was shown to occur through theubiquitin-binding function of p62 leading to aggregation ofubiquitinated caspase-8 for subsequent proteolytic autoactivationof caspase-8 [24, 25]. The present study investigated theinvolvement of p62 in cancer progression and the feasibility ofsmall molecule-mediated targeting of p62 for the therapeuticimprovement in therapy-resistant cancer cells using head andneck squamous cell carcinoma (HNSCC) as a model.HNSCC can be divided into two groups based on the status of

human papillomavirus (HPV) infection [26–28]. Although theincidence of HPV(+ ) head and neck cancer is rising, tobacco-induced carcinogenesis remains the leading cause of HNSCC[29–31]. HPV(-) HNSCC patients are less responsive to radiotherapyand have worse survival compared to HPV(+ ) HNSCC patients[32–39]. At present, the mechanism of therapy-resistance in HPV(-)HNSCC is poorly understood. In the current study we found thatan increased autophagic flux and apoptosis defects are associatedwith radioresistance in HPV(-) HNSCC, whereas HPV(+ ) HNSCC failto induce autophagic flux and readily undergo apoptotic celldeath upon radiation treatments. We also show that theexpression levels of cytosolic p62 proteins are associated withhigh grade tumors. These results suggest an association ofcytoplasmic p62-mediated autophagy with disease progressionand therapy resistance. Importantly, pharmacological activation ofthe p62 ZZ domain using synthetic small molecule ligandspromote radiation-induced cytotoxicity via apoptosis inductionin therapy-resistant HPV(-) HNSCC. We provide evidence thatcentral to p62-dependent apoptosis in HPV(-) HNSCC is its self-oligomerization along with caspase-8 to form aggresomes-likecomplexes. Taken together, our study may pave the way for thedevelopment of a multimodal treatment to activate apoptosis in ap62-specific manner in intrinsically apoptosis-resistant cells.

MATERIALS AND METHODSCell cultureHuman HNSCC cell lines UP-SCC-090 and UP-SCC-154 (established atUniversity of Pittsburg), UM-SCC-19 (University of Michigan) and WSU-HN-12 (Wayne State University) were cultured as previously described [40].

Antibodies and reagentsThe antibodies used in this study are as follows: mouse monoclonal anti-p62 (Abcam, ab56416, 1:100,000), mouse monoclonal anti-FK2 specific toUb-conjugated proteins (Millipore, 04-263, 1:1,000), rabbit polyclonal anti-LC3 (Sigma, L7543, 1: 10,000 for immunoblotting; 1:1,000 for immuno-fluorescence staining), anti-casapse-8 (Santa Cruz, sc-56070 1:2000 forimmunoblotting, 1:200 for immunofluorescence staining), rabbit polyclonalanti-GAPDH (Santa Cruz, sc-25778, 1:2,000), rabbit polyclonal anti-GAPDH(BioWorld, AP0063, 1:20,000). The following secondary antibodies wereused: alexa fluor 488 goat anti-rabbit IgG (Invitrogen, A11029, 1:200), texasred goat anti-mouse IgG (Invitrogen, T6390, 1:500), anti-rabbit IgG-HRP(Cell Signaling, 7074, 1:10,000), and anti-mouse IgG-HRP (Cell SignalingTechnology, 7076, 1:10,000). Other reagents used in this study werebafilomycin A1 (Sigma); high capacity streptavidin agarose resin (ThermoFisher Scientific).

Ionizing radiation treatment (XRT)As we previously described [41], cells were irradiated with 0, 2, 4, and 6 Gyusing a gantry-mounted Best Theratronics Gammabeam 500 with a doserate of 1 Gy/min. Irradiation was carried out at room temperature underatmospheric oxygen conditions. Delivered dose was confirmed with theuse of a Farmer chamber.

DEVDase activity assayAs we previously described [41], cells were lysed with a 0.5% NP40 lysisbuffer, and 50 μg of protein lysates was incubated with 10mmol/L Ac-DEVD-AMC substrate (Sigma) at 37 °C for 2 h. Fluorescence was detectedusing a SpectraMax Gemini (Molecular Probes, Carlsbad, CA) with 360 nmexcitation and 460 nm emission.

Establishment of p62-knockdown WSU12 cell lineScrambled shRNA sequence (shScram; catalog no. RHS4346) and threeshRNA against p62 GIPZ (shp62; clone ID no. V3LHS-375194, -375195,-375197) were obtained from Open Biosystems (Huntsville, AL). WSU12cells were transfected with shScram or p62-targeting shRNA vectors usingLipofectamine 2000 (Invitrogen) and selected with 0.25 μg/mL puromycin.The resulting pooled population were referred to as shp62-94, shp62-95,and shp62-97, respectively.

Immunofluorescence stainingHuman HNSCC cells were cultured on cover slips and fixed with 4%paraformaldehyde in PBS (pH 7.4) for 15min at room temperature. Afterwashing three times with PBS, the cells were permeabilized with 0.5%Triton X-100/PBS solution for 15min and washed three times with PBS for5 min. After three washes with PBS, the cells were incubated with blockingsolution (2% BSA in PBS) for 1 h and then with primary antibody overnightat 4 °C. Next day, the cells were washed five times for 10min each timewith PBS and then incubated with secondary antibody for 1 h. The cellswere washed five times with PBS for 10min each time, and DAPI stainedfor 10min. After three washes with PBS, the coverslips were mounted onslides using the coverslips Fluoro-GEL (Electron Microscopy Sciences).Confocal images were taken by laser scanning confocal microscope 510Meta (Zeiss) and analyzed using Zeiss LSM Image Browser (ver. 4.2.0.121).

Immunoprecipitation assayWSU12 cells, treated with vehicle control (DMSO) or YOK1104 for 12 h inthe presence or absence of XRT (6 Gy), were lysed in binding buffer [20mMTris-HCl, pH 7.6, 125mM NaCl, and 1% Nonidet P-40 with a cocktail ofprotease and phosphatase inhibitors (Roche)] through one cycle offreezing in liquid nitrogen and thawing in a 42 °C water bath. GenomicDNA was sheared by passing the extracts through a 26-gauge needle fourtimes. The resulting lysates were centrifuged at 3000 × g for 15 min topellet the cellular debris. Total 500 μg proteins were incubated with 2 μgcontrol IgG or mouse monoclonal antibody raised against full-lengthrecombinant caspase-8 of human origin (Santa Cruz, sc-56070) forovernight, followed by incubation with 40 μl of 50% protein A agarosebead slurry for 1 h. The beads were washed with the binding buffer for5 min at 4 °C with gentle agitation; washing was repeated three times. Theproteins bound to protein A/G agarose beads were dissociated in 2X SDSsample buffer, heated at 100 °C for 5 min, and separated on a SDS-PAGE.For immunoblot analysis of caspase-8 using caspase-8 IP products, thesecondary HRP antibody (GeneTex, GTX221667-01), which specificallyreacts with the native, non-reduced form of mouse IgG, was used todistinguish the caspase-8 band from immunoglobulin heavy chain bands.

p62 immunohistochemistryHNSCC blocks were retrieved from the Pathology core at Wayne StateUniversity and sections were deparaffinized and then hydrated. Microwaveheat-induced antigen retrieval was performed using the Antigen CitrusPlus Retrieval Solution (BioGenex, HK081-5K). Non-specific binding siteswere blocked with 2.5% normal horse serum in a wet chamber at 4 oC,overnight. p62 (AbCam, ab56416) was diluted 1:2000 in blocking solutionthen incubated in a wet chamber at 4 oC for 6 h. The ImmPRESS™ HRP Anti-Mouse IgG (Peroxidase) Polymer Detection Kit (Vector Laboratories, MP-7402) and the ImmPACTTM DAB Peroxidase Substrate (Vector Laboratories,SK-4105) were used to develop the signal of stained slides followed bycounterstaining with Mayer’s Hematoxylin (Scytek, HMM999). Images wereobtained using a Zeiss Axioplan 2 microscope. Scoring was performed by a

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pathologist using a scale of 0= no staining, 1= low, 2=moderate, 3=high stain.

Proximity Ligation Assay (PLA)The proximity ligation assay was performed according to the manufac-turer’s protocol using the Duolink™ In Situ Red Starter Kit Mouse/Rabbit(Sigma-Aldrich). Deparaffinization, hydration, and antigen retrieval wereperformed as described in the IHC section. p62 (AbCam, ab56416) and LC3(Cell Signaling Technology, 2775) were diluted 1:2000 in antibody diluentovernight in a humidity chamber at 4 °C. Images were obtained using aLeica DMi3000 B Fluorescence Microscope.

Statistical analysisAll experiments were repeated at least three times, and all data arepresented as the mean ± SD. Statistical analysis was performed usingStudent’s unpaired two-tailed t-test or Chi-square test whose significancewas determined according to p values (p < 0.001(***), p < 0.01(**),p < 0.05(*)).

RESULTSActivation of the autophagy–lysosome pathway is a marker ofradioresistance in HPV(-) HNSCCTo understand the molecular mechanisms of radioresistance inHNSCC cells, we assessed the relationship between autophagicactivities and radiosensitivity in HPV(-) HNSCC cells (WSU12 andUM19) compared to HPV(+ ) HNSCC cells (UP090 and UP154). TheHPV status in these cell lines was confirmed by RT-PCR analyses ofthe HPV oncogenes E6 and E7 (Supplementary Fig. 1a). Consistentwith previous reports [32–39], HPV(-) WSU12 and UM19 cells weremore resistant to radiation treatment than HPV(+ ) UP090 andUP154 cells (Fig. 1a). Upon radiation treatment, apoptotic celldeath was evident in HPV(+ ) HNSCC cells as determined bycaspase activation (Fig. 1b) whereas no apoptosis was observed inHPV(-) HNSCC cells.

Next, we examined autophagic activity by monitoring thesynthesis of LC3 and its lipidation to LC3-II. Immunoblottingshowed that the expression level and lipidation of LC3 werehigher in HPV(-) HNSCC cells in comparison with HPV(+ ) HNSCCcells (Fig. 1c), suggesting that HPV inhibits autophagy in host cellsthrough an unknown mechanism. A strong accumulation of LC3-IIwas observed in HPV(-) HNSCC cells treated with bafilomycin A1,an inhibitor of the lysosomal proton pump V-ATPase that inhibitsautophagosome–lysosome fusion (Fig. 1d, e). These resultsindicate that the increases of LC3-II in HPV(-) HNSCC cells areindeed due to enhanced autophagic flux which was not observedin HPV(+ ) HNSCC cells (Fig. 1d, e). To confirm differentialautophagic flux between HPV(-) and HPV(+ ) HNSCC in vivo, wevisualized the targeting of p62+ to LC3+ autophagic membranesby the proximity ligation assay (PLA) that detects the colocaliza-tion of two proteins within the distance of 40 nm. Usingantibodies against p62 and LC3 the PLA readily revealed anumber of p62+LC3+ autophagic membranes in HPV(-) HNSCCtissues, whereas this complex was barely detected in HPV(+ )HNSCC tissues (Fig. 1f, g). These results demonstrate that therapy-resistant HPV(-) HNSCC cells have higher levels of autophagic fluxrelative to HPV(+ ) HNSCC cells.

The cytosolic levels of p62 correlates with the progression ofHNSCCIn autophagy, cargoes are selectively recognized and collected byautophagic cargo receptors such as p62 and other Sequestosome-like receptors (SLRs) [42]. Recent studies have suggested afunctional and/or progonstic significance of cytosolic vs. nuclearp62 in tumor progression and therapeutic responses [43, 44]. Wetherefore examined the subcellular localization of p62 in HNSCCtissues. The specificity of p62 antibody was confirmed usingcontrol and p62 knockdown HPV(-) WSU cells (SupplementaryFig. 1b). The staining intensity of nuclear and cytoplasmic p62 inHNSCC tissues was evaluated by a pathologist and scored on a

Fig. 1 Defective apoptosis and increased autophagic flux in radioresistant HPV(-) HNSCC. a Clonogenic cell survival assay of HPV(+ )HNSCC cell lines (UP090 and UP154) and HPV(-) HNSCC (WSU12 and UM19) upon ionizing radiation (XRT) (*p < 0.05, ***p < 0.001). b DEVDase(caspase-3/caspase-7) activity assay in HNSCC cells at indicated time points post irradiation at 6 Gy (***p < 0.001). c Immunoblotting analysis ofLC3 or GAPDH in HPV(-) HNSCC cell lines (WSU12 and UM19) and HPV(+ ) HNSCC cell lines (UP090 and UP154). d Autophagic flux assay usingWSU12, UM19 or UP154 cells in the absence or presence of bafilomycin A (500 nM, 6 h). e Quantification of LC3-II band intensity of d. Errorbars represent means ± SD from three independent experiments (***p < 0.001, ns: non-significant). f In situ detection of autophagosomes inHPV(-) and HPV(+ ) HNSCC tissues by p62 and LC3 PLA. Scale bar: 30 μm. g The average number of positive foci in p62 and LC3 PLAquantitated at 10X magnification (n= 4, ***p < 0.001).

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scale of 0 to 3. Typical immunostaining patterns of p62 in normaltissues, HPV(-) low grade, and HPV(-) high-grade HNSCC are shownin Fig. 2a. Interestingly, cytoplasmic translocation of p62 wasassociated with disease progression within the same patient tissue(Fig. 2b). An analysis of 7 normal tissue, 9 low grade, 5 moderate,and 8 high-grade HNSCC specimens showed that p62 was almostexclusively found in the nucleus of normal tissues and low-gradeHNSCC (Fig. 2c). However, the localization of p62 gradually shiftedfrom the nucleus to the cytosol as the tumors advanced intomoderate and high-grade HNSCC (Fig. 2c, d). The overallexpression levels of p62 was also associated with higher tumorgrade (Fig. 2e). Collectively, these results showed associationbetween the cytosolic localization of p62 and HPV(-) HNSCC tumorprogression, consistent with a previous report that increased LC3puncta, high cytoplasmic p62, and low nuclear p62 expressionsare associated with poor prognosis and aggressive clinicopatho-logic features of oral squamous cell carcinoma (OSCC) [43].

Synthetic small-molecule ligands of p62 induce autophagicflux in HPV(-) HNSCC cellsWe previously developed a small-molecule ligand, YOK1104, tothe ZZ domain of p62 that induces self-polymerization of p62 andp62 interaction with LC3 on autophagic membranes [22]. Giventhe correlation between cytoplasmic p62 and HNSCC progressionand the association between autophagic flux and radioresistance,we tested whether YOK1104 can modulate radiosensitivity of HPV(-) HNSCC cells. We first confirmed that YOK1104 binds to the ZZdomain of p62 using streptavidin pulldown assay with biotiny-lated YOK1104 (Fig. 3a–d). Next, we tested whether YOK1104 canaffect p62-mediated autophagic flux in HPV(-) and HPV(+ ) HNSCCcells. Immunofluorescence analyses of HPV(-) HNSCC cells treated

with YOK1104 revealed a drastic increase in the formation ofcytosolic p62 and LC3 puncta, which showed strong colocalizationto form p62+LC3+ double positive cytosolic puncta (Fig. 3e). Theformation of p62+LC3+ puncta was associated with the increasesin the synthesis and lipidation of LC3 (Fig. 3f) as well as thecolocalization of LC3 puncta with the cytosolic puncta positive forLAMP1, a lysosomal marker (Fig. 3g). Consistent with increasedautophagic flux, YOK1104 enhanced the formation of cytosolicpuncta positive for Beclin1, indicative of enhanced autophagoso-mal nucleation (Fig. 3h). These results show that the small-molecule ligand YOK1104 increases the activity of p62 inmacroautophagy, autophagosome biogenesis, and autophagicflux in HPV(-) HNSCC cells. In contrast, YOK1104 exerted no suchautophagy-inducing activity in HPV(+ ) HNSCC cells as evidencedby the minimal levels of LC3-II (Fig. 3f). Likewise, neither theformation of cytosolic puncta positive for LC3 (Fig. 3i, j), thecolocalization of LC3+ autophagosomes with LAMP1+ lysosomes(Fig. 3k, l) nor Beclin1 (Fig. 3m) was found in HPV(+ ) HNSCC cellstreated with YOK1104. These results show that YOK1104 inducesp62-dependent autophagy in HPV(-) HNSCC but not in autophagy-defective HPV(+ ) HNSCC cells.

YOK1104 enhances radiosensitivity of therapy-resistant HPV(-) HNSCC cells via apoptosis induction in a p62-dependentmannerAccumulating evidence suggests that autophagy is associatedwith therapy-resistance [8, 9]. Besides the role of p62 inautophagy, recent studies demonstrated multi-functions of p62including its role in caspase-8 activation [24, 25]. Here, weexamined whether YOK1104 activation of p62 further increasestherapy-resistance via its induction of autophagic flux or by

Fig. 2 Cytosolic p62 is associated with advanced HNSCC. a Immunohistochemical analysis of p62 in normal tissue (top), low grade (middle),and high grade (bottom) HNSCC. Scale bar, 50 μm. b Immunohistochemical analysis of cytosolic and nuclear p62 localization corresponding totumor progression. Scale bar, 50 μm. c Frequency of nuclear p62 in normal tissues and low, moderate, and high-grade HNSCC as assessed byexpression levels (score 0–3), analyzed by Chi-square test, p= 0.025. d Frequency of cytoplasmic p62 in normal tissues and low, moderate, andhigh-grade HNSCC as assessed by expression levels (score 0–3), analyzed by Chi-square test, p < 0.001. e Frequency of overall p62 in normaltissues and low, moderate, and high-grade HNSCC as assessed by expression levels in both nucleus and cytoplasm (score 0–3), analyzed byChi-square test, p= 0.001.

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converting its signal to programmed cell death. To this end, wefirst determined the effects of YOK1104 treatments onradiotherapy-induced cytotoxicity in HNSCC cells. YOK1104rendered intrinsically radio-resistant HPV(-) UM19 and WSU12cells sensitive to ionizing radiation (Fig. 4a, b). Notably, the radio-sensitizing efficacy of YOK1104 on HPV(-) WSU12 and UM19 cellswas achieved at a dose as low as 2 Gy, a clinically relevant dose.Unlike HPV(-) HNSCC cells, YOK1104 had little effect on survival ofautophagy-defective HPV(+ ) UP090 and UP154 cells (Fig. 4a).Moreover, the efficacy of YOK1104 was higher in UM19 cells withhigher autophagic flux compared to WSU12 cells (Fig.1d andSupplementary Fig. 1c–e). These results suggest a positivecorrelation between the ability of YOK1104 to promote

radiotoxicity and the levels of cells’ intrinsic autophagic flux. Toconfirm that YOK1104-mediated cytotoxicity is dependent of p62,WSU12 cells were engineered to stably express shRNA against p62(Fig. 4c). As shown in Fig. 4b, p62 knockdown virtually abolishedthe ability of YOK1104 to enhance radiotoxicity, demonstratingthe specificity of YOK1104 to p62.Next, we determined whether YOK1104-enhanced radiotoxicity

in HPV(-) HNSCC cells involves apoptotic cell death. NeitherYOK1104 treatment nor ionizing radiation up to 6 Gy alone wassufficient to activate DEVDase, reflecting activation of pro-caspases 3 and 7 (Fig. 4d). However, combined treatments withradiation and YOK1104 induced caspase activity, which waseffectively counteracted by p62 knockdown (Fig. 4d). These results

Fig. 3 p62 ligand YOK1104 induces autophagy in radioresistant HPV(-) HNSCC. a, b Chemical structures of free or biotinylated p62-ZZsmall-molecule ligand YOK1104. c Schematic illustration of full-length and ZZ domain-deleted mutant p62 constructs. d In vitro YOK1104-Biotin pulldown assay using full-length p62-GFP or ΔZZ-p62-GFP proteins, purified from HEK293 cells after transient transfection of thoseexpression vectors. e Immunostaining of p62 (red) and LC3 (green) in WSU12 HPV(-) HNSCC treated with vehicle control (DMSO) or 5 μMYOK1104. Scale bar: 10 μm. f Immunoblot analysis of p62, LC3, or GAPDH with or without YOK1104 treatment. g Immunostaining of LC3 (red)and LAMP1 (green) in WSU12 HPV(-) HNSCC treated with vehicle control (DMSO) or 5 μM YOK1104. Scale bar: 10 μm. h Immunostaining ofBeclin1 (red) in UM19 HPV(-) HNSCC treated with vehicle control (DMSO) or 5 μM YOK1104. Scale bar: 20 μm. i Immunostaining of p62 (red)and LC3 (green) in UP090 HPV(+ ) HNSCC treated with vehicle control (DMSO) or 5 μM YOK1104. Scale bar: 10 μm. j Quantification ofcolocalization (yellow) of p62 with LC3 in e and i (n= 20 cells) (***p < 0.001, ns non-significant). k Immunostaining of LC3 (red) and LAMP1(green) in UP154 HPV(+ ) HNSCC treated with vehicle control (DMSO) or 5 μM YOK1104. Scale bar: 10 μm. l Quantification of colocalization(yellow) of LC3 with LAMP1 in g and k (n= 20 cells) (***p < 0.001, ns: non-significant). m Immunostaining of Beclin1 (red) in UP154 HPV(+ )HNSCC treated with vehicle control (DMSO) or 5 μM YOK1104. Scale bar: 20 μm.

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suggest that YOK1104-activated p62 induces apoptotic cell deathin intrinsically therapy-resistant and apoptosis-defective HNSCCcells upon ionizing radiation treatment.

YOK1104-activated p62 induces the formation of aggresome-like caspase-8 complexWe investigated whether pharmacological activation of p62induces apoptotic cell death through the modulation of autopha-gic flux and/or caspase-8 in HPV(-) HNSCC cells upon irradiation.As expected, YOK1104 treatment enhanced autophagic flux inHPV(-) HNSCC cells as indicated by increased LC3 lipidation in thepresence of bafilomycin A1 (Fig. 5a–d, lanes 4 vs. 3). It should benoted that immunoblots with shorter exposure time are shown toexamine the accumulation of LC3-II and p62 in the presence ofbafilomycin following treatments with YOK1104 and/or radiation.Strikingly, such autophagic flux was drastically reduced byirradiation (Fig. 5a–d, lanes 6 vs. 5). YOK1104-induced LC3-II andp62 accumulations, detected by bafilomycin treatment, wasdecreased upon irradiation (Fig. 5a–d, lanes 8 vs. 4). This indicatesthat the autophagy system is downregulated by co-treatment withYOK1104 and radiation. Thus, the p62 ZZ ligand YOK1104-promoted radiotoxicity is unlikely due to its sudden induction ofautophagic flux at a toxic level.Caspase-8 is an initiator caspase, pivotal in the extrinsic

pathway of apoptosis [45, 46]. Death ligand binding to its cognatereceptors on the plasma membrane induces the formation of the

death-inducing signaling complex (DISC) where caspase-8 isrecruited and activated [45, 46]. The E3 ligase Cullin3, that isassociated with DISC, polyubiquitinates caspase-8 which subse-quently interacts with the UBA domain of p62. These interactionspromote aggregation and full activation of caspase-8. Subse-quently, p62 transports the activated caspase-8 to cytosolicubiquitin-rich aggresomes, which in turn induce apoptoticsignaling pathways [25, 47]. Here, we examined whetherYOK1104-activated p62 in combination with ionizing radiationactivates the ubiquitinated caspase-8, similarly to caspase-8activation in the extrinsic pathway. Immunoblot analyses detectedan active (cleaved) form of caspase-8 in apoptosis-prone HPV(+ )UP154 cells upon irradiation (Supplementary Fig. 2a), but not inapoptosis-resistant HPV(-) WSU12 cells (Fig. 5e). While YOK1104treatment alone had no effect on caspase-8 activation, co-treatment with radiation resulted in caspase-8 activation in HPV(-) HNSCC cells (Fig. 5e). Moreover, caspase3/7 activity induced byco-treatment was abolished in the presence of caspase-8 inhibitor(Fig. 5f). These results indicate the functional significance ofcaspase-8 as an initiator of caspase cascade upon YOK1104 andradiation treatments.Next, we monitored the ubiquitination of caspase-8 in HPV(-)

WSU12 cells treated with irradiation and/or YOK1104. Theubiquitination of caspase-8 was prominent upon irradiation alonebut not by YOK1104 (Fig. 5g, right panel). A control experiment forthe specificity of caspase-8 immunoprecipitation is shown in

Fig. 4 YOK1104-mediated p62 activation promotes radiotoxicity in HPV(-) via caspase activation in a p62-dependent manner. aClonogenic cell survival assay of HPV(-) HNSCC cell line UM19 and HPV(+ ) lines (UP090 and UP154) in the absence or presence of 5 μMYOK1104 at the indicated radiation doses (*p < 0.05, **p < 0.01, ns non-significant). b Clonogenic cell survival assay of control and p62 KDWSU12 cells in the absence or presence of 5 μM YOK1104 at indicated radiation doses. c Immunoblot analysis of p62 in HPV(-) WSU12 cellstransfected with control (Scramble) or three independent vectors targeting p62 mRNA (sh p62-(#94, #95, #97). d Caspase-3 (DEVDase)-likeactivity was measured in control and p62 KD (#94, #95, #97) WSU12 cells treated with 5 μM YOK1104 and/or 6 Gy XRT (*p < 0.05, ns non-significant).

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Supplementary Fig. 2b. We then asked whether YOK1104-boundp62 facilitates the formation of caspase-8 aggresomes in HPV(-)HNSCC cells after irradiation. Immunostaining analyses of caspase-8 and p62 did not detect caspase-8 aggresomes when treated

with either irradiation or YOK1104 alone (Fig. 5h). Strikingly, alarge number of caspase-8 aggresomes were generated by co-treatment with radiation and YOK1104 (Fig. 5h). Moreover, wedetected colocalization of caspase-8 with K63-linked ubiquitin

Fig. 5 YOK1104-activated p62 sequesters ubiquitinated caspase-8 into aggresome-like structures in response to irradiation. aAutophagic flux assay using WSU12 cells treated with 5 μM YOK1104 (12 h) and/or 500 nM bafilomycin A1 (6 h) with or without irradiation at6 Gy. b Quantification of LC3-II band intensity of a. Error bars represent means ± SD from three independent experiments (***p < 0.001, ns non-significant). c Autophagic flux assay using UM19 cells treated with 5 μM YOK1104 (12 h) and/or 500 nM bafilomycin A1 (6 h) with or withoutirradiation at 6 Gy. d Quantification of LC3-II band intensity of c. Error bars represent means ± SD from three independent experiments (**p <0.01, ***p < 0.001, ns non-significant). e Immunoblot analysis of caspase-8 of WSU12 cells in the presence or absence of 5 μM YOK1104 with orwithout irradiation at 6 Gy. f Caspase-3/7 (DEVDase)-like activity was measured in WSU12 cells treated with 5 μM YOK1104 and 6 Gy XRT in theabsence or presence of caspase-8 inhibitor (20 μM, 25 h). ***p < 0.001. g Immunoblot analyses of ubiquitinated proteins and caspase-8 usingtotal WSU12 cell lysates (left panel) or caspase-8 immunoprecipitates (right panel) after treatment with 5 μM YOK1104 with or withoutirradiation at 6 Gy. h Immunostaining of caspase-8 (green) and p62 (red) in WSU12 HPV(-) HNSCC treated with vehicle control (DMSO) or 5 μMYOK1104 with or without irradiation at 6 Gy. Scale bar: 10 μm. i Immunostaining of caspase-8 (green) and K63-linked ubiquitin (red) in UM19HPV(-) HNSCC treated with vehicle control (DMSO) or 5 μM YOK1104 with or without irradiation at 6 Gy. Scale bar: 10 μm. j Analysis ofsubcellular localization of caspase-8 (green) using a single focal plane at the middle of the nucleus (DAPI, blue). k Immunostaining of caspase-8 (green) in control and p62 KD WSU12 HPV(-) HNSCC treated with 5 μM YOK1104 and irradiation at 6 Gy. Scale bar: 10 μm.

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chains, further supporting the formation of caspase-8+ aggre-somes by co-treatment with YOK1104 and radiation (Fig. 5i).Confocal microscopy revealed that caspase-8 puncta, generatedafter YOK1104/radiation co-treatment, were present mostly in thecytoplasm including the perinuclear region (Fig. 5j). Importantly,the formation of caspase-8 puncta was abolished when p62 wasdownregulated in WSU12 cells (Fig. 5k). Immunofluorescencestaining analyses showed that while treatment with YOK1104alone increased LC3+p62+ puncta, co-treatment with YOK1104and radiation reduced LC3+p62+ puncta and increased caspase-8+p62+ puncta (Supplementary Fig. 2c–e). Taken together, theseresults demonstrate that while YOK1104 promotes autophagicflux, co-treatment with radiation exerts its cytotoxic effect byreducing autophagic flux and, in parallel, inducing apoptotic celldeath mediated by caspase-8 and p62.In conclusion, this study suggests that irradiation induces

ubiquitination of caspase-8 by an as-yet-unknown mechanism,and ubiquitination of caspase-8 itself is insufficient to interact withp62 for the formation of aggresome. Importantly, pharmacologicactivation and oligomerization of p62 provides a focal point forcaspase-8 aggregation leading to caspase-8 activation (Fig. 6).Thus, we propose that small-molecule ligands to the p62 ZZdomain have therapeutic potential as radiosensitizers in intrinsi-cally apoptosis-resistant HPV(-) HNSCC.

DISCUSSIONIn this study, we investigated the mechanisms underlyingradioresistance in HPV(-) HNSCC and examined the potential ofp62 as a druggable target to improve the efficacy of radiationtherapy. To this end, we first characterized the distinct responsesof HPV(+ ) vs. HPV(-) HNSCC to ionizing radiation and observedthat the latter were not only less susceptible to radiotoxicity butalso resistant to caspase activation. HPV(-) HNSCC exhibited bothdrastically greater levels of autophagosomes and their acceleratedturnover. In association with this, our study suggests that thecytosolic level of p62 and cellular level of autophagy flux correlate

with the radioresistance and the progression of HPV(-) HNSCC. Weexplored whether this pro-survival autophagic pathway can betargeted by novel therapies. A small-molecule ligand to the ZZdomain of p62, termed YOK-1104, was successfully used to inducep62 self-oligomerization, autophagic sequestration of its cargoes,and autophagosome biogenesis in HPV(-), in sharp contrast to HPV(+ ) HNSCC. Such structural and functional activation of p62 by asynthetic ligand sensitized HPV(-) HNSCC to radiation-inducedradiotoxicity. Mechanistically, irradiation of HPV(-) HNSCC resultsin ubiquitination of caspase-8, which appears to be a prerequisitebut insufficient for its interaction with p62. Importantly, pharma-cologic activation of p62 facilitates caspase-8+p62+ aggresome-like structures for the activation of the subsequent apoptoticsignaling cascade. Our finding that synthetic ligand-mediated p62activation promotes radiotoxicity via induction of apoptosis incells with high autophagic flux is particularly significant since thisapproach may induce malignant cancer-specific cell death withless normal cell toxicity.Interestingly, HPV(-) HNSCC cells not only exhibited higher basal

degrees of autophagy flux and p62–LC3 interaction as opposed totheir HPV(+ ) counterparts, but also were more readily responsiveto p62-ZZ ligand-mediated autophagy induction. Our resultsshowed that synthetic activation of p62 via its ZZ domain primesHPV(-), but not HPV(+ ), HNSCC for radiation-induced cell deathby sequestering ubiquitinated caspase-8 into aggresome-likestructures. Failure of p62 activation in HPV(+ ) cells would hinderthe self-oligomeric activity of p62 and the ability to sequester itscargoes, including caspase-8. Given the significance of p62-regulated ER stress signaling in the regulation of both autophagyand programmed cell death, it would be of importance toinvestigate functional relationship between radiosensitivity andmisregulation of p62 and/or the N-degron pathway in the future.Interestingly, photodynamic therapy (PDT) directed at the ER/mitochondria using benzoporphyrin derivative (BPD) synergizedwith YOK1104 for caspase activation whereas PDT using NPe6, aselective photosensitizer for lysosomes, failed to do so (Supple-mentary Fig 3).Upon death signals, the E3 ligase Cullin3 polyubiquitinates

autoprocessed caspases-8 catalytic domain fragments, which inturn recruits p62 to the ubiquitinated chains. p62 transports theliberated catalytic domain of casepase-8 to ubiquitin-rich “aggre-somes” in the cytosol [25]. A pertinent question in our study is whyubiquitinated caspase-8 fails to form aggresomes with p62 in HPV(-) HNSCC cells upon irradiation. We surmise that in the absence ofadaptor proteins such as FADD in DISC, monomers of caspase-8and p62 fail to interact with each other in the formation ofaggresome. Importantly, our study demonstrated that YOK1104-induced oligomerization and activation of p62 are sufficient toinitiate the formation of caspase-8+p62+ puncta upon co-treatment with radiation even in the absence of death receptoractivation. It would be of importance to determine the subcellularlocation where YOK1104-activated p62 oligomers initiate aggre-some formation with caspase-8+ upon irradiation. It also remainsto be seen how p62-mediated aggregation of caspase-8 activatesdownstream executioner caspases. One possibility is that p62-associated caspases-8+ aggresomes prolong the half-life ofpolyubiquitinated caspase-8 beyond the necessary threshold forthe activation of downstream executioner caspases, instead ofbeing targeted to the proteasome, as previously suggested [25].Taken together, our study provides molecular insights into thefunctional interplay between autophagy and programmed celldeath pathways regulated by p62. We, thus, propose that p62 is adruggable target for the manipulation of sensitivity to deathstimuli in cancer cells, typically resistant to both intrinsic andextrinsic cell death stimuli (Supplementary Fig. 4). In addition tocancer therapeutics, our findings may have broad biomedicalapplications since imbalance between autophagy and apoptosisare often involved in many human diseases.

Fig. 6 A graphical illustration of p62-dependent caspase-8activation upon irradiation. Synthetic p62-ZZ ligands, such asYOK1104, induce p62 self-oligomerization, its interaction with LC3,and autophagosome biogenesis. Ionizing radiation treatmentinduces ubiquitination of caspase-8. A synthetic ligand-activatedp62 via its ZZ domain promotes aggregation of ubiquitinatedcaspase-8 into aggresomes-like structures leading to apoptosis.

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DATA AVAILABILITYAll data is available in the manuscript file and its supplementary information file oravailable upon request.

REFERENCES1. Gump JM, Thorburn A. Autophagy and apoptosis: what is the connection? Trends

Cell Biol. 2011;21:387–92.2. Cecconi F, Levine B. The role of autophagy in mammalian development: cell

makeover rather than cell death. Dev Cell. 2008;15:344–57.3. Tuzlak S, Kaufmann T, Villunger A. Interrogating the relevance of mitochondrial

apoptosis for vertebrate development and postnatal tissue homeostasis. GenesDev. 2016;30:2133–51.

4. Thorburn A. Apoptosis and autophagy: regulatory connections between twosupposedly different processes. Apoptosis. 2008;13:1–9.

5. Singh R, Letai A, Sarosiek K. Regulation of apoptosis in health and disease: thebalancing act of BCL-2 family proteins. Nat Rev Mol Cell Biol. 2019;20:175–93.

6. Mizushima N, Komatsu M. Autophagy: renovation of cells and tissues. Cell.2011;147:728–41.

7. Kung CP, Budina A, Balaburski G, Bergenstock MK, Murphy M. Autophagy in tumorsuppression and cancer therapy. Crit Rev Eukaryot Gene Expr. 2011;21:71–100.

8. Mathew R, Karantza-Wadsworth V, White E. Role of autophagy in cancer. Nat RevCancer. 2007;7:961–7.

9. Thorburn A, Thamm DH, Gustafson DL. Autophagy and cancer therapy. MolPharm. 2014;85:830–8.

10. Wu H, Che X, Zheng Q, Wu A, Pan K, Shao A, et al. Caspases: a molecular switchnode in the crosstalk between autophagy and apoptosis. Int J Biol Sci.2014;10:1072–83.

11. Pattingre S, Tassa A, Qu X, Garuti R, Liang XH, Mizushima N, et al. Bcl-2 anti-apoptotic proteins inhibit Beclin 1-dependent autophagy. Cell. 2005;122:927–39.

12. Rubinsztein DC, Codogno P, Levine B. Autophagy modulation as a potentialtherapeutic target for diverse diseases. Nat Rev Drug Discov. 2012;11:709–30.

13. Mulcahy Levy JM, Thorburn A. Autophagy in cancer: moving from understandingmechanism to improving therapy responses in patients. Cell Death Differ.2020;27:843–57.

14. Xu R, Ji Z, Xu C, Zhu J. The clinical value of using chloroquine or hydroxy-chloroquine as autophagy inhibitors in the treatment of cancers: a systematicreview and meta-analysis. Medicine. 2018;97:e12912.

15. Bachmair A, Finley D, Varshavsky A. In vivo half-life of a protein is a function of itsamino-terminal residue. Science. 1986;234:179–86.

16. Tasaki T, Sriram SM, Park KS, Kwon YT. The N-end rule pathway. Annu Rev Bio-chem. 2012;81:261–89.

17. Kwon YT, Kashina AS, Davydov IV, Hu RG, An JY, Seo JW, et al. An essential role ofN-terminal arginylation in cardiovascular development. Science. 2002;297:96–99.

18. Sriram SM, Kwon YT. The molecular principles of N-end rule recognition. NatStruct Mol Biol. 2010;17:1164–5.

19. Kwon YT, Xia Z, Davydov IV, Lecker SH, Varshavsky A. Construction and analysis ofmouse strains lacking the ubiquitin ligase UBR1 (E3alpha) of the N-end rulepathway. Mol Cell Biol. 2001;21:8007–21.

20. Tasaki T, Kwon YT. The mammalian N-end rule pathway: new insights into itscomponents and physiological roles. Trends Biochem Sci. 2007;32:520–8.

21. Cha-Molstad H, Sung KS, Hwang J, Kim KA, Yu JE, Yoo YD, et al. Amino-terminalarginylation targets endoplasmic reticulum chaperone BiP for autophagythrough p62 binding. Nat Cell Biol. 2015;17:917–29.

22. Cha-Molstad H, Yu JE, Feng Z, Lee SH, Kim JG, Yang P, et al. p62/SQSTM1/Sequestosome-1 is an N-recognin of the N-end rule pathway which modulatesautophagosome biogenesis. Nat Commun. 2017;8:102.

23. Cha-Molstad H, Lee SH, Kim JG, Sung KW, Hwang J, Shim SM, et al. Regulation ofautophagic proteolysis by the N-recognin SQSTM1/p62 of the N-end rule path-way. Autophagy. 2018;14:359–61.

24. Ullman E, Pan JA, Zong WX. Squamous cell carcinoma antigen 1 promotescaspase-8-mediated apoptosis in response to endoplasmic reticulum stress whileinhibiting necrosis induced by lysosomal injury. Mol Cell Biol. 2011;31:2902–19.

25. Jin Z, Li Y, Pitti R, Lawrence D, Pham VC, Lill JR, et al. Cullin3-based poly-ubiquitination and p62-dependent aggregation of caspase-8 mediate extrinsicapoptosis signaling. Cell. 2009;137:721–35.

26. Attner P, Du J, Nasman A, Hammarstedt L, Ramqvist T, Lindholm J, et al. The roleof human papillomavirus in the increased incidence of base of tongue cancer. IntJ Cancer. 2010;126:2879–84.

27. Chaturvedi AK, Engels EA, Anderson WF, Gillison ML. Incidence trends for humanpapillomavirus-related and -unrelated oral squamous cell carcinomas in theUnited States. J Clin Oncol. 2008;26:612–9.

28. Hammarstedt L, Lindquist D, Dahlstrand H, Romanitan M, Dahlgren LO, JonebergJ, et al. Human papillomavirus as a risk factor for the increase in incidence oftonsillar cancer. Int J Cancer. 2006;119:2620–3.

29. Kobayashi K, Hisamatsu K, Suzui N, Hara A, Tomita H, Miyazaki T. A review of HPV-related head and neck cancer. J Clin Med. 2018;7:241.

30. Gillison ML, Chaturvedi AK, Anderson WF, Fakhry C. Epidemiology of humanpapillomavirus-positive head and neck squamous cell carcinoma. J Clin Oncol.2015;33:3235–42.

31. Jethwa AR, Khariwala SS. Tobacco-related carcinogenesis in head and neckcancer. Cancer Metastasis Rev. 2017;36:411–23.

32. Ang MK, Patel MR, Yin XY, Sundaram S, Fritchie K, Zhao N, et al. High XRCC1protein expression is associated with poorer survival in patients with head andneck squamous cell carcinoma. Clin Cancer Res. 2011;17:6542–52.

33. Fakhry C, Westra WH, Li S, Cmelak A, Ridge JA, Pinto H, et al. Improved survival ofpatients with human papillomavirus-positive head and neck squamous cell car-cinoma in a prospective clinical trial. J Natl Cancer Inst. 2008;100:261–9.

34. Hong AM, Dobbins TA, Lee CS, Jones D, Harnett GB, Armstrong BK, et al. Humanpapillomavirus predicts outcome in oropharyngeal cancer in patients treatedprimarily with surgery or radiation therapy. Br J Cancer. 2010;103:1510–7.

35. Sethi S, Ali-Fehmi R, Franceschi S, Struijk L, van Doorn LJ, Quint W, et al. Char-acteristics and survival of head and neck cancer by HPV status: a cancer registry-based study. Int J Cancer. 2012;131:1179–86.

36. Lill C, Kornek G, Bachtiary B, Selzer E, Schopper C, Mittlboeck M, et al. Survival ofpatients with HPV-positive oropharyngeal cancer after radiochemotherapy issignificantly enhanced. Wien Klin Wochenschr. 2011;123:215–21.

37. Mellin H, Friesland S, Lewensohn R, Dalianis T, Munck-Wikland E. Human papil-lomavirus (HPV) DNA in tonsillar cancer: clinical correlates, risk of relapse, andsurvival. Int J Cancer. 2000;89:300–4.

38. Sedaghat AR, Zhang Z, Begum S, Palermo R, Best S, Ulmer KM, et al. Prognosticsignificance of human papillomavirus in oropharyngeal squamous cell carcino-mas. Laryngoscope. 2009;119:1542–9.

39. Worden FP, Kumar B, Lee JS, Wolf GT, Cordell KG, Taylor JM, et al. Chemoselectionas a strategy for organ preservation in advanced oropharynx cancer: responseand survival positively associated with HPV16 copy number. J Clin Oncol.2008;26:3138–46.

40. Jung YS, Najy AJ, Huang W, Sethi S, Snyder M, Sakr W, et al. HPV-associateddifferential regulation of tumor metabolism in oropharyngeal head and neckcancer. Oncotarget. 2017;8:51530–41.

41. Cho WJ, Kessel D, Rakowski J, Loughery B, Najy AJ, Pham T, et al. Photodynamictherapy as a potent radiosensitizer in head and neck squamous cell carcinoma.Cancers. 2021;13:1193.

42. Stolz A, Ernst A, Dikic I. Cargo recognition and trafficking in selective autophagy.Nat Cell Biol. 2014;16:495–501.

43. Liu JL, Chen FF, Lung J, Lo CH, Lee FH, Lu YC, et al. Prognostic significance of p62/SQSTM1 subcellular localization and LC3B in oral squamous cell carcinoma. Br JCancer. 2014;111:944–54.

44. Wang Y, Zhang N, Zhang L, Li R, Fu W, Ma K, et al. Autophagy regulates chromatinubiquitination in DNA damage response through elimination of SQSTM1/p62.Mol Cell. 2016;63:34–48.

45. Peter ME, Krammer PH. The CD95(APO-1/Fas) DISC and beyond. Cell Death Differ.2003;10:26–35.

46. Ashkenazi A, Dixit VM. Death receptors: signaling and modulation. Science.1998;281:1305–8.

47. Gonzalvez F, Lawrence D, Yang B, Yee S, Pitti R, Marsters S, et al. TRAF2 sets athreshold for extrinsic apoptosis by tagging caspase-8 with a ubiquitin shutofftimer. Mol Cell. 2012;48:888–99.

ACKNOWLEDGEMENTSWe thank the members of the Kim and Kwon laboratories for their critical discussions.

AUTHOR CONTRIBUTIONSS.H.L., W.J.C, Y.T.K., and H-R.C.K. designed the experiments, interpreted the results,and prepared the manuscript. S.H.L. and W.J.C performed the majority of theexperiments. A.J.N., A-D.S., T.P., and W.S. contributed to p62 immunohistochemistryand A.J.N., A-D.S., and T.P. performed proximity ligation assay as well as theinterpretation of the results. W.J.C., J.R., B.L., W.K.C., and H.E.K. contributed to ionizingradiation treatment (XRT). C.H.J. performed immunoprecipitation assays andinterpreted the results. H-R.C.K., Y.T.K., W.S., and H.E.K. provided guidance, specializedreagents, and expertise.

FUNDINGThis research was supported by NIH grants CA 123362 (H-R.C.K.) and DE023181 (IK,H-R.C.K and HEK), Korean National Science Foundation Brain Pool Program Award (H-R.C.K.), the International Research & Development Program of the National Research

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Foundation of Korea (NRF) funded by the Ministry of Science and ICT(2019K1A3A1A12070180 to S.H.L.), NRF grant funded by the Korea government(MSIT) (No. NRF-2020R1A5A1019023 to Y.T.K.), and Basic Science Research Programthrough NRF funded by the Ministry of Education (NRF-2021R1A2B5B03002614 to Y.T.K.), and SNU Hospital (to Y.T.K.).

ETHICS APPROVALCollection and utilization of tissue specimens were approved by the InstitutionalReview Board (IRB) of Wayne State University.

COMPETING INTERESTSThe authors declare no competing interests.

ADDITIONAL INFORMATIONSupplementary information The online version contains supplementary materialavailable at https://doi.org/10.1038/s41419-021-04301-7.

Correspondence and requests for materials should be addressed to Yong Tae Kwonor Hyeong-Reh C. Kim.

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